A new type of Eucommia ulmoides fermentation liquid and its application in large yellow croaker farming

Abstract

The invention discloses a novel eucommia fermentation liquid, which is obtained by inoculating activated bacillus subtilis into a culture medium containing eucommia leaf powder for fermentation after expansion culture, wherein the culture medium containing eucommia leaf powder mainly consists of eucommia leaf powder and water, and the mass fraction of eucommia leaf powder is 25-30%. The invention also discloses the application of the eucommia fermentation liquid, especially the application of the eucommia fermentation liquid as an additive in the preparation of large yellow croaker feed. It has been proved by experiments that the novel eucommia fermentation liquid provided by the invention can not only significantly promote the growth of large yellow croaker, but also significantly improve the body color and muscle quality of large yellow croaker.

Description

Technical Field

[0001] The invention relates to aquaculture additives and applications thereof, in particular to a novel eucommia fermentation liquid and also to the application of the eucommia fermentation liquid in large yellow croaker farming. Background Art

[0002] Large yellow croaker (Larimichthys crocea) belongs to the Perciformes, Sciaenidae, and Larimichthys genus. It is favored by consumers for its delicious meat and high nutritional value. After breaking through the technical bottleneck of seedling cultivation, the large yellow croaker industry has developed rapidly, and the aquaculture output has increased year by year. In 2021, it has exceeded 240,000 tons, ranking first in marine fish aquaculture output. It is an important aquaculture species in my country's coastal areas and has extremely high economic value.

[0003] However, as the large yellow croaker farming industry develops rapidly, problems such as insufficient growth-promoting feed performance and declining quality of farmed fish are becoming increasingly prominent; at the same time, consumers are paying more and more attention to the quality of large yellow croaker, and wild large yellow croaker with golden color and firm meat is always in short supply. In order to shorten the breeding cycle and improve the quality of farmed fish, farmers have to use iced fish to make pulp for feeding, and even worse, they use banned drugs, which seriously wastes fishery resources and endangers food safety.

[0004] Eucommia ulmoides Oliv, also known as cotton, Si Zhong, and silk cotton wood, is a tree species of the Eucommia family and the genus Eucommia. It is one of the unique economic forest tree species in my country, and there is only one of its kind in the world. It is native to the mountainous areas of Guizhou, Sichuan, and Hunan in my country. After introduction and cultivation, it can grow and develop normally in the northern regions with an annual average temperature of 11℃ to 18℃ and a minimum temperature of more than -20℃. Traditionally, my country's use of Eucommia resources has been limited to the development and utilization of Eucommia bark in medical care. Further research has found that the Eucommia leaves with the largest output contain high levels of active substances such as alkaloids, polysaccharides, aucubin, flavonoids, and chlorogenic acid, and have great potential for development and utilization. Many studies have shown that Eucommia leaves have significant effects on promoting growth, anti-inflammatory, antibacterial, inhibiting obesity, and improving meat quality, but the utilization efficiency is low when directly added to feed.

[0005] Fermentation of Chinese herbal medicines can not only reduce or remove toxic and harmful substances by using microorganisms, but also promote the release of effective ingredients and increase the content of related active substances by using enzymes produced by microorganisms. Eucommia leaves are a byproduct of Eucommia cultivation. When mixed with water, they become viscous. When the mass fraction exceeds 5%, it is difficult to maintain stable stirring and ventilation, and it blocks a variety of pure liquid fermentation tanks. Current research mostly uses solid-state fermentation alone or after mixing with feed raw materials, which is inefficient. The liquid fermentation technology of Chinese herbal medicines can effectively improve the release efficiency of active ingredients, and the fermentation process is more controllable. Therefore, in order to promote the efficient utilization of Eucommia leaves and improve the growth and quality of farmed large yellow croaker, the development of a new type of Eucommia fermentation liquid using liquid fermentation technology is of great significance to promote the healthy development of the industry. Summary of the invention

[0006] One of the purposes of the present invention is to provide a novel eucommia fermentation liquid which can effectively improve the growth and muscle quality of artificially cultured large yellow croaker.

[0007] The object of the present invention is achieved through the following technical scheme: the novel eucommia fermentation liquid is obtained by inoculating activated Bacillus subtilis into a culture medium containing eucommia leaf powder for fermentation after expansion culture, wherein the culture medium containing eucommia leaf powder mainly consists of eucommia leaf powder and water, and the mass fraction of eucommia leaf powder is 25-30%.

[0008] The fermentation conditions of the present invention are preferably: rotation speed 200-300r, ventilation volume 6-8vv, and fermentation 30-40h.

[0009] As an embodiment of the present invention, in a 100L fermentation tank, 25kg of Eucommia leaf powder and 45kg of purified water were added, 25L of bacterial solution was transferred into a 50L tank, the rotation speed was 250r, the ventilation volume was 7vv, and the fermentation was carried out for 36h.

[0010] Activation of Bacillus subtilis: Bacillus subtilis powder was inoculated into complete culture medium and cultured at 200r / min for 6h for activation.

[0011] Furthermore, the complete culture medium used for strain activation contains 6.25 g of tryptone, 6.25 g of hydrolyzed casein peptone, 10 g of beef extract, 5 g of dry yeast powder, 2 g of diammonium hydrogen citrate, 1.53 g of K2HPO4, 5 g of NaAc, 0.58 g of MgSO4·7H2O, 0.19 g of MnSO4, 1 ml of Tween-80, and 1 L of distilled water, with a pH of 6.5-7.0, and is sterilized by high-pressure steam.

[0012] Expansion culture of Bacillus subtilis: inoculate 1L of cultured bacterial liquid into a 50L fermenter, with 25L of medium in the fermenter. Rotate at 355r, temperature at 3730.5℃, dissolved oxygen content >40%, adjust pH to 6.5-7.5 with ammonia water. Continue culture for 8h.

[0013] Furthermore, the method for preparing the culture medium for expanding the culture of the strain is as follows: 10g of peptone, 10g of corn starch, 10g of glucose, 10g of molasses mother liquor, 20g of yeast extract, 7.5g of Na2HPO4·12H2O, 3.2g of (NH4)2SO4, 1g of CaCO3, 0.05g of defoaming agent, and 1L of sterile water; the above materials except glucose are added to the fermentation tank, and then steam is passed and sterilized at 120°C for 30min; the pH is adjusted to 7.430.1 with NaOH; the glucose is sterilized at 110°C for 30 minutes and then added to the fermentation tank; the fermentation temperature is 36±1°C; the fermentation pH is 7.030.1, which is adjusted with ammonia water.

[0014] In the present invention, after the fermentation is completed, the mixture is firstly kept at 80°C for 20 to 30 minutes for pasteurization, and then filtered under aseptic conditions to obtain the Eucommia fermentation liquid.

[0015] Another object of the present invention is to provide a method for preparing the novel Eucommia ulmoides fermentation broth.

[0016] Specifically, the activated Bacillus subtilis is expanded and cultured, and then inoculated into a culture medium containing eucommia leaf powder for fermentation. The culture medium containing eucommia leaf powder mainly consists of eucommia leaf powder and water, and the mass fraction of eucommia leaf powder is 25-30%.

[0017] The fermentation conditions of the present invention are preferably: rotation speed 200-300r, ventilation volume 6-8vv, and fermentation 30-40h.

[0018] As an embodiment of the present invention, in a 100L fermentation tank, 25kg of Eucommia leaf powder and 45kg of purified water were added, 25L of bacterial solution was transferred into a 50L tank, the rotation speed was 250r, the ventilation volume was 7vv, and the fermentation was carried out for 36h.

[0019] Activation of Bacillus subtilis: Bacillus subtilis powder was inoculated into complete culture medium and cultured at 200r / min for 6h for activation.

[0020] Furthermore, the complete culture medium used for strain activation contains 6.25 g of tryptone, 6.25 g of hydrolyzed casein peptone, 10 g of beef extract, 5 g of dry yeast powder, 2 g of diammonium hydrogen citrate, 1.53 g of K2HPO4, 5 g of NaAc, 0.58 g of MgSO4·7H2O, 0.19 g of MnSO4, 1 ml of Tween-80, and 1 L of distilled water, with a pH of 6.5-7.0, and is sterilized by high-pressure steam.

[0021] Expansion culture of Bacillus subtilis: inoculate 1L of cultured bacterial liquid into a 50L fermenter, with 25L of medium in the fermenter. Rotate at 355r, temperature at 3730.5℃, dissolved oxygen content >40%, adjust pH to 6.5-7.5 with ammonia water. Continue culture for 8h.

[0022] Furthermore, the method for preparing the culture medium for expanding the culture of the strain is as follows: 10g of peptone, 10g of corn starch, 10g of glucose, 10g of molasses mother liquor, 20g of yeast extract, 7.5g of Na2HPO4·12H2O, 3.2g of (NH4)2SO4, 1g of CaCO3, 0.05g of defoaming agent, and 1L of sterile water; the above materials except glucose are added to the fermentation tank, and then steam is passed and sterilized at 120°C for 30min; the pH is adjusted to 7.430.1 with NaOH; the glucose is sterilized at 110°C for 30 minutes and then added to the fermentation tank; the fermentation temperature is 36±1°C; the fermentation pH is 7.030.1, which is adjusted with ammonia water.

[0023] In the present invention, after the fermentation is completed, the mixture is firstly kept at 80°C for 20 to 30 minutes for pasteurization, and then filtered under aseptic conditions to obtain the Eucommia fermentation liquid.

[0024] Another object of the present invention is to provide an application of the Eucommia ulmoides fermentation broth.

[0025] Specifically, it relates to the application of the novel Eucommia ulmoides fermentation liquid as an additive in the preparation of large yellow croaker feed.

[0026] Another object of the present invention is to provide an aquaculture feed comprising the Eucommia ulmoides fermentation liquid.

[0027] Specifically, the aquaculture feed includes basic feed, fish oil and the above-mentioned Eucommia ulmoides fermentation liquid; wherein the Eucommia ulmoides fermentation liquid accounts for 4% of the total mass, and the fish oil accounts for 5.5-6.5%.

[0028] The basic feed includes white fish meal, krill meal, soybean meal, soybean protein concentrate, high-gluten flour, choline chloride, soybean lecithin powder, Vc phosphate, calcium dihydrogen phosphate, vitamin premix, mineral premix, attractant, mildew inhibitor and antioxidant, etc.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The novel Eucommia ulmoides fermentation liquid provided by the present invention is obtained by a liquid fermentation method and uses a liquid culture medium containing more than 30% solid matter. After the fermentation is completed, the filtrate obtained by filtration can be directly applied to feed without going through a concentration and purification process.

[0031] (2) The novel Eucommia ulmoides fermentation liquid provided by the present invention can not only significantly improve the growth and muscle quality of large yellow croaker, but also significantly increase the redness value of the side of large yellow croaker fry after adding the novel Eucommia ulmoides fermentation liquid to the feed, although it significantly reduces the brightness and yellowness of the side of large yellow croaker fry. From the test results, the redness and yellowness values ​​of the abdomen show an increasing trend, which is beneficial to improving the economic value of large yellow croaker. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the effect of the new Eucommia ulmoides fermented liquid on the survival rate of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0033] Figure 2 Figure 4 Effect of the new Eucommia ulmoides fermented liquid on the final body weight of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0034] Figure 3 Figure 4 Effect of the new Eucommia ulmoides fermented liquid on the weight gain rate of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0035] Figure 4 This is the effect of the new Eucommia ulmoides fermented liquid on the specific growth rate of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0036] Figure 5 Figure 4 Effect of the new Eucommia ulmoides fermented liquid on the body length of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0037] Figure 6 This is the effect of the new Eucommia ulmoides fermented liquid on the fatness of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0038] Figure 7 This is the effect of the new Eucommia ulmoides fermented liquid on the lateral brightness (L*) of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0039] Figure 8 This is the effect of the new Eucommia ulmoides fermented liquid on the red value (a*) of the side of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0040] Fig. 9 This is the effect of the new Eucommia ulmoides fermented liquid on the yellow value (b*) of the side of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0041] Fig.10 This is the effect of the new Eucommia ulmoides fermented liquid on the abdominal brightness (L*) of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0042] Fig.11 This is the effect of the new Eucommia ulmoides fermented liquid on the abdominal red value (a*) of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0043] Fig.12 This is the effect of the new Eucommia ulmoides fermented liquid on the yellow value (b*) of the side of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0044] Fig.13 This is the effect of the new Eucommia ulmoides fermented liquid on the muscle hardness of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0045] Fig.14 This is the effect of the new Eucommia ulmoides fermented liquid on the muscle viscosity of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0046] Fig.15This is the effect of the new Eucommia ulmoides fermented liquid on the muscle elasticity of juvenile large yellow croaker. “*”, “**” and “***” all indicate significant differences, “*” indicates P<0.05, “**” indicates P<0.01, and “***” indicates P<0.001.

[0047] Fig.16 This is the effect of the new Eucommia ulmoides fermentation liquid on the muscle adhesion of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, "**" indicates P<0.01, and "***" indicates P<0.001.

[0048] Fig.17 This is the effect of the new Eucommia ulmoides fermentation liquid on the muscle adhesion of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, "**" indicates P<0.01, and "***" indicates P<0.001.

[0049] Fig.18 This is the effect of the new Eucommia ulmoides fermented liquid on the chewiness of the muscle of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, "**" indicates P<0.01, and "***" indicates P<0.001.

[0050] Fig.19 This is the effect of the new Eucommia ulmoides fermentation liquid on the muscle recovery of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, "**" indicates P<0.01, and "***" indicates P<0.001. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods, but does not constitute any limitation to the present invention.

[0052] Unless otherwise specified, the reagents used in the present invention can be purchased from commercial sources.

[0053] 1. Screening of fermentation conditions for Eucommia ulmoides leaf powder

[0054] 1.1 Small-scale experiment

[0055] 1.1.1 Single Factor Experiment

[0056] Under the conditions of fermentation temperature of 36°C, fermentation time of 48h, inoculation amount of 10%, rotation speed of 180r and pH value of 6.0, the effect of water content on the release content of effective ingredients in Eucommia ulmoides leaves was studied. The results are shown in Table 1.

[0057] Table 1 Effect of water content on the release of active ingredients in Eucommia ulmoides leaves

[0058]

[0059] Table 1 Effect of water content on the release of active ingredients in Eucommia ulmoides leaves

[0060]

[0061] 1.1.2 Orthogonal test

[0062] In order to further determine the optimal process parameters for releasing the active ingredients in Eucommia ulmoides leaves by fermentation, the fermentation temperatures (A) were selected as 30°C, 33°C, and 36°C; the inoculation amounts (B) were 2%, 4%, 6%, 8%, 10%, and 12%; and the fermentation times (C) were 24h, 36h, 48h, 72h, and 96h as factors and levels. A three-factor orthogonal design was carried out with a total of 30 treatments. The results are shown in Tables 2 and 3.

[0063] Table 2 Results of orthogonal experiment on chlorogenic acid content

[0064]

[0065] Table 2 Results of orthogonal experiment on chlorogenic acid content

[0066]

[0067] Table 2 Results of orthogonal experiment on chlorogenic acid content

[0068]

[0069] Table 3 Results of all active ingredient contents in orthogonal experiment

[0070]

[0071] Table 3 Results of all active ingredient contents in orthogonal experiment

[0072]

[0073] 1.1.3 Optimal combination conditions

[0074] The results of the single factor experiment show that when the moisture content is 70-75%, the difference between the chlorogenic acid release content of the fermented Eucommia leaf powder and the unfermented Eucommia leaf powder is the largest. This indicates that under this moisture content, the increase in the chlorogenic acid release content is due to the action of Bacillus. In addition, the reduction of moisture and Eucommia polysaccharide content will significantly reduce the cost of the spray drying part, which is the most expensive part in actual production.

[0075] From the orthogonal experiment variance analysis, it can be seen that the optimal combination is A2B4C4, that is, the fermentation temperature is 33℃, the inoculation amount is 8%, the fermentation time is 72h, the rotation speed is 180r, and the pH value is 6.0. Under the optimal level combination conditions of each factor, the verification test of the release of chlorogenic acid from the fermented Eucommia leaf powder was carried out, and the OD value of chlorogenic acid was measured to be 630, which is higher than any treatment in the orthogonal experiment, proving that the test results are credible. This result is also close to the chlorogenic acid release of the unfermented Eucommia leaf powder with a water content of 90%, indicating that when the content is reduced, it does not rely on the osmotic effect of water on the chlorogenic acid in the cell wall, but relies on the enzymatic wall-breaking effect of Bacillus and the promotion of the synthesis of chlorogenic acid precursors to increase the release of chlorogenic acid, thereby improving the release efficiency and reducing the actual production cost.

[0076] 2. Preparation of Eucommia ulmoides fermentation broth

[0077] According to the optimal conditions obtained from the small-scale experiment in triangular flasks: fermentation temperature 33°C, inoculation amount 8%, fermentation time 72h, rotation speed 180r, pH value 6.0, combined with the actual conditions of the fermentation equipment in the pilot fermentation, the pilot-scale production was carried out after optimizing the combination.

[0078] 2.1 Bacterial Activation

[0079] Under sterile conditions, 10 g of Bacillus subtilis powder was directly inoculated into 1 L of complete culture medium and cultured at 200 r / min for 6 h for activation.

[0080] The method of preparing the culture medium for strain activation is as follows: 6.25 g of tryptone, 6.25 g of hydrolyzed casein peptone, 10 g of beef extract, 5 g of dry yeast powder, 2 g of diammonium hydrogen citrate, 1.53 g of K2HPO4, 5 g of NaAc, 0.58 g of MgSO4·7H2O, 0.19 g of MnSO4, and 1 ml of Tween-80, add to 1 L of distilled water, and adjust the pH to 6.5-7.0. Sterilize with high pressure steam.

[0081] 2.2 Expansion of fermentation tank strains

[0082] Inoculate 1L of cultured bacterial liquid into a 50L fermenter, with 25L of culture medium in the fermenter. RPM 355r, temperature 3730.5℃, dissolved oxygen >40%, adjust pH to 6.5-7.5 with ammonia water. Continue to culture for 8h.

[0083] The fermentation tank strain expansion medium is prepared as follows: 10g peptone, 10g corn starch, 10g glucose, 10g molasses mother liquor, 20g yeast extract, 7.5g Na2HPO4·12H2O, 3.2g (NH4)2SO4, 1g CaCO3, 0.05g defoamer, and 1L of sterile water. After adding to the fermentation tank, steam is passed and sterilized at 120℃ for 30min; pH is adjusted to 7.430.1 with NaOH; after glucose is sterilized at 110℃ for 30 minutes, the medium is added; the fermentation temperature is 3631℃; the fermentation pH is 7.030.1, which is adjusted with ammonia water.

[0084] 2.3 Fermentation tank experiment

[0085] In a 100L fermentation tank, 20kg of Eucommia leaf powder and 45kg of purified water were added, 25L of bacterial solution was transferred from a 50L tank, the rotation speed was 250r, the ventilation volume was 7vv, and the fermentation was carried out for 36h. After the fermentation was completed, the mixture was first pasteurized at 80℃ for 20-30min, and then filtered under sterile conditions to obtain the Eucommia fermentation liquid.

[0086] 3. Application of the new Eucommia ulmoides fermentation liquid

[0087] The basic feed formula for large yellow croaker fry is: 40% white fish meal, 3% krill meal, 10% peeled soybean meal, 24.65% high-gluten flour, 6% soy protein concentrate, 0.2% choline chloride, 2% soy lecithin powder, 0.05% Vc phosphate, 2% monocalcium phosphate, 1% vitamin premix, 0.5% mineral premix, 0.5% attractant, 0.05% mildew inhibitor and 0.05% ethoxyquin.

[0088] The preparation process of large yellow croaker fry feed is as follows: white fish meal, krill meal, peeled soybean meal, high-gluten flour and soybean protein concentrate are mixed and then ultra-finely crushed to 120 meshes to obtain mixture 1; choline chloride, soybean lecithin powder, Vc phosphate, calcium dihydrogen phosphate, vitamin premix, mineral premix, attractant, mildew inhibitor and ethoxyquinoline are mixed to obtain mixture 2; mixture 1, mixture 2 and fish oil are mixed and then 10-15% of water is added to granulate.

[0089] Comparative feed 1

[0090] The basic feed for juvenile large yellow croaker uses fish oil as the oil source, which accounts for 6% of the total feed mass. No new type of Eucommia ulmoides fermentation liquid is added, and 4% microcrystalline cellulose is used instead.

[0091] Example Feed 2

[0092] Fish oil and new Eucommia ulmoides fermented liquid are added to the basic feed of large yellow croaker fry, the addition amount of the new Eucommia ulmoides fermented liquid is 4% of the total weight of the basic feed of large yellow croaker fry, and the fish oil accounts for 6% of the total weight of the feed.

[0093] The following table 3 is the formula of the above examples and comparative examples:

[0094] Table 1 Feed formula of embodiment and comparative example

[0095]

[0096] Among them, vitamin premix (IU or mg kg -1 Dry weight): Vitamin A palmitate, 3000000 IU; Vitamin D3, 1200000 IU; DL-α-tocopherol 40.0 mg kg -1 ; Menadione, 8.0 mg kg -1 ; Thiamine-HCl, 5.0 mg kg -1 ; Riboflavin, 5.0 mg kg-1; D-calcium pantothenate, 16.0 mg kg-1; Pyridoxine-HCl, 4.0 mg kg-1; Inositol, 200.0 mg kg -1 ; D-biotin, 8.0 mg kg -1 ; Folic acid, 1.5 mg kg -1 ; p-aminobenzoic acid, 5.0 mg kg -1 ; Niacin, 20.0 mg kg -1 ; Vitamin B12, 0.01 mg kg -1 ; Vitamin C (content 35%), 2000.0 mg kg -1 . Mineral premix (mg kg -1 Dry weight) Ca(H2PO4)·H2O, 675.0; CoSO4·H2O, 0.15; CuSO4·H2O, 5.0; FeSO4·7H2O, 50.0; KCl, 0.1; MnSO4·2H2O, 101.7; MnSO4·2H2O, 18.0; NaCl, 80.0; NaSeO3·H2O, 0.05; ZnSO4·7H2O, 20.0. The attractant was prepared by mixing 1% betaine and 3% glycine with a filler (all provided by Qingdao Master Biotechnology Co., Ltd.)

[0097] Experimental example: Effects of adding new Eucommia ulmoides fermented liquid on growth and body color of juvenile large yellow croaker

[0098] A total of 240 large yellow croakers with an initial body weight of 12.930.02 g were selected for the experiment and randomly divided into 6 groups with three replicates in each group and 40 fish in each replicate for a 63-day feeding and growth experiment; the culture water temperature was 18-22°C.

[0099] The mice were fed with feed 1 as the CON group, and the mice were fed with feed 2 as the DZ group.

[0100] Growth and body shape were measured directly using an electronic balance and vernier caliper; muscle texture was measured using a texture analyzer.

[0101] Statistical and analytical methods:

[0102] Survival rate (SR, %) = Wa / Wb × 100%

[0103] Weight gain rate (WG, %) = (Na-Nb) / Nb×100%

[0104] Fullness (CFR, g / cm 3 %) = Na / (La) 3 ×100%

[0105] Specific growth rate (SGR, % / day) = (lnNa-lnNb) / experimental period × 100%

[0106] Where Wa is the number of yellow croaker fry in each breeding bucket after the end of breeding, Wb is the initial number of fry in each breeding bucket; La is the final body length; Na and Nb are the final body weight and initial body weight, respectively.

[0107] The experimental data will be statistically analyzed using one-way analysis of variance in SPSS 20.0, and Tukey's multiple comparisons will be performed after significant differences, with a significance level of P < 0.05. The experimental data obtained are expressed as means 3 standard errors (means 3S.EM, n = 3).

[0108] 1) Growth performance

[0109] like Figure 1-4 As shown in the results, the addition of the new Eucommia fermented liquid to the feed did not affect the survival rate of yellow croaker fry, but significantly increased the final weight, weight gain rate and specific growth rate of yellow croaker fry. This shows that adding 4% of the new Eucommia fermented liquid to the basic feed can significantly promote the growth of yellow croaker fry without affecting the survival rate, effectively shortening the breeding cycle.

[0110] 2) Body shape

[0111] like Figure 5-6 As shown in the results, the addition of the new Eucommia fermented liquid to the feed did not affect the body length and fatness of the yellow croaker fry, and the addition showed a trend of increasing the body length and reducing the fatness. This shows that adding 4% of the new Eucommia fermented liquid to the basic feed will not cause abnormal body shape and obesity in yellow croaker fry.

[0112] 3)Body color

[0113] like Figure 7-12As shown in the figure, although the addition of the new Eucommia fermented liquid to the feed significantly reduced the brightness and yellowness of the side of the large yellow croaker fry, it significantly increased the redness value of the side; the red and yellow values ​​of the abdomen showed an upward trend. This shows that adding 4% of the new Eucommia fermented liquid to the basic feed can significantly improve the body color of large yellow croaker fry.

[0114] 4) Muscle texture

[0115] like Figure 13-19 As shown in the results, the addition of the new Eucommia fermented liquid to the feed did not affect the muscle hardness, viscosity, elasticity, cohesion, adhesion and recovery of large yellow croaker, but significantly improved the muscle chewiness. This shows that the addition of 4% of the new Eucommia fermented liquid to the basic feed can significantly improve the muscle quality of large yellow croaker fry.

Claims

1. New Eucommia ulmoides fermentation liquid, Its characteristics are: In a 100L fermentation tank, 20kg of Eucommia leaf powder, 45kg of purified water, 25L of bacterial liquid, a rotation speed of 250r, and a ventilation volume of 7vv were used for 36h fermentation; after the fermentation, it was firstly kept at 80℃ for 20-30min for pasteurization, and then filtered under aseptic conditions to obtain Eucommia fermentation liquid; the bacterial liquid was obtained by expanding the culture of activated Bacillus subtilis.

2. According to the novel Eucommia ulmoides fermentation liquid of claim 1, Its characteristics are: Activation of the Bacillus subtilis: directly inoculate 10 g of Bacillus subtilis powder into 1 L of complete culture medium under sterile conditions, and culture at 200 r / min for 6 h for activation; The strain activation culture medium is prepared by: 6.25 g of tryptone, 6.25 g of hydrolyzed casein peptone, 10 g of beef extract, 5 g of dry yeast powder, 2 g of diammonium hydrogen citrate, and K 2 HPO 4 1.53 g, NaAc 5 g, MgSO 4 •7H 2 O 0.58g, MnSO 4 0.19g, Tween-80 1ml, dilute to 1L distilled water, adjust pH to 6.5~7.0; sterilize by high pressure steam.

3. According to the novel Eucommia ulmoides fermentation liquid of claim 1, Its characteristics are: The expansion culture of the Bacillus subtilis is as follows: 1L of the cultured bacterial liquid is inoculated into a 50L fermenter, and the fermenter medium is 25L; the fermentation conditions are: rotation speed 355r, temperature 37±0.5°C, dissolved oxygen>40%, pH adjusted to 6.5-7.5 with ammonia water, and continuous culture for 8h.

4. According to the novel Eucommia ulmoides fermentation liquid of claim 3, Its characteristics are: The preparation method of the culture medium for expanding the culture of Bacillus subtilis is as follows: 10g of peptone, 10g of corn starch, 10g of glucose, 10g of molasses mother liquor, 20g of yeast extract, 2 HPO 4 12H 2 O 7.5g, (NH 4 ) 2 SO 4 3.2 g, CaCO 3 1g, defoaming agent 0.05g, and sterile water to 1L; add the above materials except glucose into the fermentation tank, and then pass steam and sterilize at 120℃ for 30min; adjust the pH to 7.4±0.1 with NaOH; after glucose is sterilized at 110℃ for 30 minutes, add it into the fermentation tank.

5. Use of the Eucommia ulmoides fermentation liquid according to any one of claims 1 to 4 as an additive in preparing large yellow croaker feed.

6. Aquaculture feed containing Eucommia ulmoides fermented liquid, including basic feed, fish oil, Its characteristics are: Contains the eucommia fermentation broth according to any one of claims 1 to 4.

7. The aquaculture feed comprising Eucommia ulmoides fermentation liquid according to claim 6, Its characteristics are: The eucommia fermentation liquid accounts for 4% of the total mass of the feed, and the fish oil accounts for 6% of the total mass of the feed.

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